Single Molecule Manipulation: Edge-Cutting Techniques for Studies of Biomolecular Machinery
Ming Li
Institute of Physics, Chinese Academy of Sciences

The past decade has witnessed rapid development of single-molecule techniques. They can address numerous biological questions, which were not accessible via ensemble measurements. In this talk, I will first give a brief introduction of two widely used single-molecule manipulation techniques, i.e., the optical tweezers and the magnetic tweezers. I then give two examples to show how these edge-cutting techniques have led to new insights on the dynamics and reaction mechanisms of enzymes. The first example is about the processive motion of kinesins that transport cargos by walking unidirectionally along microtubule tracks. The molecular machines respond to their surroundings with great flexibility, using thermal noise positively for their functions. They are very different from man-made machines that operate at energies much higher than thermal fluctuations. The second example to which I will pay more attention concerns the mechanisms of helicases that unwind double-stranded DNA (dsDNA). Two models have been proposed for them. The passive model deals with helicases which unwind dsDNA by trapping unwinding fluctuations of the dsDNA while they are translocating on the single-stranded tails of the dsDNA. The passive helicases also harness thermal fluctuation energies. However, an active helicase interact strongly with the dsDNA so that the thermal fluctuations are not very important. E. coli UvrD is such an active helicase. It must dimerize on the DNA before it can unwind it. Its unwinding rate decreases when the force acting to separate the two strands of DNA is increased. The results lead to a strained-inchworm mechanism in which a conformational change that bends and tenses the ssDNA is required to activate the dimer.

Bio:
Prof Ming Li earned his Ph.D. in physics from the University of Wuerzburg, Germany in 1998. He then conducted postdoctoral research in the University of Illinois at Chicago before he joined the Institute of Physics, Chinese Academy of Sciences (CAS) in 2001. He won the "National Distinguished Young Scholar Award" in 2003. He is now a professor of physics and the director of the Laboratory of Soft Matter Physics in the Institute of Physics, CAS. His current research interests include single-molecule biological physics and nanomaterials physics.

Measurement of the Interaction between Microtubule-associated Proteins and Microtubules by Optical Tweezers
Chun-Hua Xu
Institute of Physics, Chinese Academy of Sciences

Cytoskeleton is protein fibrous structure in eukaryotic cells, including microtubules, actin filaments and intermediate filaments. They, together with their associated proteins, constitute a dynamic system in cells. Microtubule-associated proteins (MAPs) regulate the organization, dynamics and function of the microtubules. They can even change the mechanical properties of the microtubules. AtMAP65-1 is a new type of MAPs found in Arabidopsis. It can induce the formation of microtubule bundles by forming cross-bridges between the microtubules. It has been reported that microtubule bundles induced by AtMAP65-1 are sensitive to salt concentration in biochemical experiments. We measured the unbinding force between the microtubules and AtMAP65-1 by a home-made dual-optical tweezers apparatus. In order to get the kinetic parameters, we fitted the force histograms based on the Bell-Evans-model of multiple bonds. Under a force loading rate of 9 pN/s, the most probable unbinding force for the single bond is calculated to be 14.7 ± 0.8 and 16.8 ± 0.8 pN for samples with and without a 100 mM NaCl treatment, respectively. The obtained intrinsic dissociation rate constants reveal that NaCl shortens the natural lifetime of the bond by 50%, and lowers the activation barrier by 3.4%. It is obvious that NaCl can weaken the combination between MAPs and microtubules. The results help to explain how salt concentration regulates microtubule arrays via AtMAP65-1 during the interphase of cells.

Microsphere Fabrication via Microfluidic Approaches
Xiuqing Gong
Nano Science and Technology Program, The Hong Kong University of Science and Technology

Microfluidic method, directing the flow of microliter volumes along microscale channels, offers the advantages of precise control of reagent loading, fast mixing and an enhanced reaction rate, cessation of the reaction at specific stages, and more. Basically, there are two microfluidic flow regimes, continuous flow and segmented flow (suspended droplets, channel-spanning slug, and wall-wetting films). Both flow regimes offer chemical reaction applications. e.g., continuous flow formation of polymer nanospheres and inorganic nanoparticles, size- and shape-control synthesis by segmented flow, and precipitate-forming reactions in droplets, wherein the segmented flow has gained more popularity in that area. The compartmentalization of segmented flow offers many advantages to chemical reactions. Here, we report the microfluidic fabrication of magnetically responsive microspheres, macroporous polymer microspheres and hollow titania microspheres.